γ-Aminobutyric acid (GABA) was originally synthesized in 1883, however it was not until 1950 that it was identified to be an integral part of the mammalian central nervous system (Petroff, 2002). The brain and spinal cord contain an abundance of GABA, whereas trivial amounts are found in peripheral tissue such as the liver, spleen and heart. GABA is synthesized by the alpha-decarboxylation of glutamate by the enzyme L-glutamic acid decarboxylase (GAD) and its cofactor pyridoxal phosphate (Petroff, 2002). GABA is an inhibitory neurotransmitter responsible for mediating the preponderance of inhibition within the brain. Three distinct forms of the GABA receptor exist; GABAA receptors which mediate fast synaptic neurotransmission (Sieghart and Sperk, 2002; Rudolph and Mohler, 2004), GABAB receptors responsible for mediating slow metabotropic actions (Couve et al., 2000; Bettler and Tiao, 2006), and GABAC receptors which mediate fast synaptic current in the retina (Bormann and Feigenspan, 1995).
1.8.1GABAA RECEPTORS
GABAA receptors (GABAARs) are members of the ligand-gated ion- channel superfamily, which also includes nicotinic acetylcholine receptors, 5- hydroxytryptamine 3 receptors and glycine receptors (Unwin, 1989; Barnard et al., 1998). GABAARs are heteropentamers with each available subunit containing a large extracellular amino-terminal domain, four transmembrane domains (TM), a large intracellular domain between TM3 and TM4, and a smaller extracellular carboxy-terminal domain. The large amino-terminus serves as the binding site for GABA as well as other compounds such as benzodiazepines, and the large intracellular loop provides a site for receptor modulation via protein interactions and post-translational modifications (Jacob et al., 2008). At this time 18 different GABAAR subunits have been identified and divided into seven different subunit classes, some with additional members: α (1 – 6), β (1 – 3), γ (1 – 3), δ, ε (1 – 3), θ, and π.
While many different subunit combinations are possible, the majority of functional GABAARs are comprised of two α subunits, two β subunits and one γ subunit; although the γ subunit can be replaced by the δ, ε, θ, or π subunit (McKernan and Whiting, 1996; Rudolph and Mohler, 2004). The large amino- terminus of the subunit regulates the assembly of particular subunits. Unassembled or misfolded proteins, as well as homomeric subunits are degraded in the endoplasmic reticulum (ER) (Gorrie et al., 1997; Bedford et al., 2001). Receptors that survive the ER are trafficked to the Golgi apparatus where they are packaged into vesicles, transported and inserted into the plasma
membrane. Several factors aid in the translocation of receptors out of the ER, including GABAA receptor-associated protein (GABARAP) which interacts with the intracellular domain of the γ subunit (Wang et al., 1999), and N-
ethylmaleimide-sensitive factor (NSF) a protein involved in intracellular vesicle fusion (Kittler et al., 2001; Zhao et al., 2007). Once inserted into the plasma membrane the β (1 – 3) and /or γ2 subunit of the GABAAR directly bind to a particular subunit of a clathrin-adaptor protein 2 (AP2) complex. This interaction initiates the clathrin-dependent endocytosis, the major mechanism by which receptors are internalized and recycled (Kittler et al., 2000).
GABAA receptor subunits are ubiquitously expressed throughout the brain, however the one focus of this document is on GABAARs within the dentate gyrus. Within this region of the adult hippocampus GABAAR subunits α1, α2, α4, β3, γ2, and δ are most highly expressed, followed by the β1subunit, α3, α5, β2 and γ3 subunits are weakly expressed, and nearly undetectable levels of γ1 subunit are observed (Sperk et al., 1997). Expression of these subunits is also developmentally regulated not only within the dentate gyrus but also throughout the hippocampus and other brain regions (Laurie et al., 1992). GABAAR subunits are targeted to different regions along the plasma membrane where they impart distinct physiological and pharmacological properties, including agonist affinity.
1.8.2FUNCTION OF HIPPOCAMPAL GABAA RECEPTORS
Upon invasion of an action potential, presynaptic GABA is released in high concentrations into the synaptic cleft where it binds to the postsynaptic GABAA
receptor. Binding of the ligand allows for negatively charged Cl- ions to flow through the channel pore and into the cell. The increase in Cl- ions allows for the inside of the cell to become more negatively charged and the cell hyperpolarizes. This hyperpolarization increases the threshold for an action potential and ultimately reduces neurotransmission.
As previously stated the majority of inhibitory neurotransmission is mediated by the GABAA receptor, and the particular subunit composition of the receptor imparts distinct characteristics. Synaptically located GABAARs mediate fast phasic inhibition (Rudolph and Mohler, 2004), whereas extrasynaptic receptors mediate tonic inhibition (Brunig et al., 2002). In addition to influencing the localization of the receptor the subunit composition dramatically influences receptor pharmacology. GABAARs are modulated by a variety of compounds including benzodiazepines, barbiturates and neurosteroids. The BZD sensitivity of the GABAAR has been well studied, particularly in relation to seizure disorders, and will be discussed in detail in Chapter 3. Generally, seizures may result from a reduction in GABAAR-mediated inhibition in the brain (Jacob et al., 2008), thus understanding the precise manner in which these receptors regulate neurotransmission is essential.
1.8.3PHASIC INHIBITION
The presynaptic release of GABA yields high, millimolar concentrations within the synaptic cleft (Mody et al., 1994) which binds to the synaptically located GABAARs and produces an inhibitory post-synaptic current (IPSC).
IPSCs regulate the rapid and precise, and near-synchronous, phasic inhibition. Studies have demonstrated that the high concentration of GABA within the cleft dissipates, either through re-uptake or diffusion, with a time constant of 100 – 500 µs (Overstreet et al., 2000; Mozrzymas et al., 2003; Mozrzymas, 2004). As opposed to mass quantities, GABA can also be released by a single synaptic vesicle. This spontaneously occurring event, results in a miniature inhibitory post- synaptic current (mIPSC). Phasic inhibition is typically mediated by GABAARs composed of α1, 2, 3 or 5 subunits together with β and γ subunits (Rudolph and Mohler, 2004). Most notable for synaptically located GABAARs is the presence of the γ2 subunit, which is essential for the synaptic clustering of GABAARs (Essrich et al., 1998).
1.8.4TONIC INHIBITION
In addition to the well-characterized rapid phasic inhibition, it is recognized that ionotropic receptors may also mediate a slower form of transmission (Mody, 2001), including a tonic activation of the receptor. As opposed to synaptically located GABAARs that interact with millimolar concentrations of GABA, the extrasynaptic receptors must have a higher affinity for GABA, as the concentration in the extracellular space is likely within the micromolar range (Nyitrai et al., 2006). In addition to their high affinity, extrasynaptic receptors must demonstrate little desensitization (Mtchedlishvili and Kapur, 2006; Glykys and Mody, 2007), a process by which receptor channels close despite the continued presence of agonist bound to the receptor. The tonic activation of extrasynaptic
GABAARs would be significantly reduced if the receptors became desensitized to the sustained presence of extrasynaptic GABA. Tonic inhibition is generally mediated by GABAARs composed of α4 or 6 subunits, together with β and δ subunits (Barnard et al., 1998). Most notable for extrasynaptic receptors is the presence of the δ subunit, which is exclusively located in the extrasynaptic membrane (Nusser et al., 1998; Wei et al., 2003).